Lysozyme antigenicity and tissue fixation.

The preservation of lysozyme (LZM) antigenicity was studied in paraffin embedded tissue blocks. The reactivity for LZM varied with the type of tissue studied, the fixative used, the osmolarity and pH of the fixative, fixation time and temperature, and the method of dehydration. In both rat and human tissues aqueous fixatives were superior to nonaqueous fixatives in retaining LZM antigenicity. Brief fixation in fixatives of low osmolarity enhanced LZM staining in the parenchymatous tissues but diminished staining in human cartilage; prolonged fixation in fixatives of high osmolarity gave opposite results. Least affected by fixation was the LZM antigenicity in the serous cells of the glands of the respiratory tract. These cells also stained most intensely for LZM of all autopsy material studied.     

Lysozyme antigenicity and tissue fixation

Summary The preservation of lysozyme (LZM) antigenicity was studied in paraffin embedded tissue blocks. The reactivity for LZM varied with the type of tissue studied, the fixative used, the osmolarity and pH of the fixative, fixation time and temperature, and the method of dehydration. In both rat and human tissues equeous fixatives were superior to nonaqueous fixatives in retaining LZM antigenicity. Brief fixation in fixatives of low osmolarity enhanced LZM staining in the parenchymatous tissues but diminished staining in human cartilage; prolonged fixation in fixatives of high osmolarity gave opposite results. Least affected by fixation was the LZM antigenicity in the serous cells of the glands of the respiratory tract. These cells also stained most intensely for LZM of all autopsy material studied.Studies supported by grants from the Sigrid Jusélius Foundation and Finska Läkaresällskapet     

The influence of fixatives and other variations in tissue processing on nuclear morphometric features

The influence on the area and numerical density of nuclei was investigated in 5-mm-thick slices of guinea pig liver for different fixatives and variations in tissue processing: delay in fixation, air drying, degree of acidity of 10% formalin (= 4% formaldehyde), Bouin and mercury-formalin fixatives, acetone and ethanol dehydration and understretching and overstretching of the paraffin-embedded sections. Air drying (either forced or as a result of delayed fixation), the type of fixative and the degree of acidity affected the nuclear area. Regarding the latter, nuclear area was approximately 25% lower for pH less than or equal to 3 as compared with pH greater than 5. In comparison with the standard tissue processing used, the nuclear density was higher after all of the variations studied (air drying, acetone dehydration and fixation). These findings indicate that nuclear area, in contrast to other tissue components, is relatively insensitive to variations in tissue processing. However, it is essential to regularly measure the pH of the fixative: deviations from pH = 7 should be carefully avoided in order to keep nuclear area variations as a result of tissue processing within acceptable limits.     

The search for an optimal DNA, RNA, and protein detection by in situ hybridization, immunohistochemistry, and solution-based methods

Clinical trials and correlative laboratory research are increasingly reliant upon archived paraffin-embedded samples. Therefore, the proper processing of biological samples is an important step to sample preservation and for downstream analyses like the detection of a wide variety of targets including micro RNA, DNA and proteins. This paper analyzed the question whether routine fixation of cells and tissues in 10% buffered formalin is optimal for in situ and solution phase analyses by comparing this fixative to a variety of cross linking and alcohol (denaturing) fixatives. We examined the ability of nine commonly used fixative regimens to preserve cell morphology and DNA/RNA/protein quality for these applications. Epstein-Barr virus (EBV) and bovine papillomavirus (BPV)-infected tissues and cells were used as our model systems. Our evaluation showed that the optimal fixative in cell preparations for molecular hybridization techniques was "gentle" fixative with a cross-linker such as paraformaldehyde or a short incubation in 10% buffered formalin. The optimal fixatives for tissue were either paraformaldehyde or low concentration of formalin (5% of formalin). Methanol was the best of the non cross-linking fixatives for in situ hybridization and immunohistochemistry. For PCR-based detection of DNA or RNA, some denaturing fixatives like acetone and methanol as well as "gentle" cross-linking fixatives like paraformaldehyde out-performed other fixatives. Long term fixation was not proposed for DNA/RNA-based assays. The typical long-term fixation of cells and tissues in 10% buffered formalin is not optimal for combined analyses by in situ hybridization, immunohistochemistry, or--if one does not have unfixed tissues--solution phase PCR. Rather, we recommend short term less intense cross linking fixation if one wishes to use the same cells/tissue for in situ hybridization, immunohistochemistry, and solution phase PCR.     

Evaluation of tissue preparation methods and paired immunofluorescence staining for immunocytochemistry of lymphomas

Eight cross-linking fixatives were tested for preservation of extracellular or intracellular IgG, IgA, IgM, IgD, kappa and lambda light chains, J chain and secretory component. Most of the selected fixatives have been used in recent immunohistochemical studies of lymphoproliferative processes and comprised routine formalin, glutaraldehyde(1%)-formalin, Baker's formalin-calcium, formalin-sublimate, acetic acid(2%)-formalin-saline, Bouin's fluid, Susa fixative, and carbodiimide. The results obtained in artificial test substrates with defined amounts of IgG or IgA and in biological substrates (colon mucosa, tonsils, and different types of lymphomas) were compared by immunofluorescence with the antigenic preservation afforded by fixation in cold 96% ethanol (with or without inclusion of a pre-fixation 48 h washing period). An antigen concentration at least an eight-fold higher was necessary for detection with most other fixatives. Bouin's and Susa fixatives were peculiar in that they required antigen concentration 150 times higher for detection of IgG but only 3-8 times higher for IgA. Light chains were relatively well preserved by all fixatives except glutaraldehyde. For all cross-linking fixatives, the extent of antigenic masking depended on the concentration of environmental proteins, and the efficiency of unmasking with pronase or trypsin, therefore, varied with the location in the tissue. The J chain was particularly vulnerable to degradation during proteolytic treatment. The extensive masking of extracellular immunoglobulin in formalin-fixed tissue afforded a relatively good signal-to-noise ratio for immunoglobulin-producing cells when kappa and lambda chains were traced. Thus, differentiation between polyclonal and monoclonal B-cell processes on the basis of cytoplasmic labelling was often better in undigested sections. However, the light-chain type of membrane immunoglobulin could usually not be determined in directly fixed tissue. Ethanol fixation preceded by washing in saline afforded such determination and also preserved certain T-cell and HLA-DR antigens as well as diffuse alpha-naphthylbutyrate esterase. Reactive and malignant macrophages could further be traced by their cytoplasmic expression of L1 antigen, both in formalin- and ethanol-fixed material.     

Immunocytochemistry in cytology. Comparative evaluation of different techniques

Immunocytochemistry represents an important tool in diagnostic cytology. Its application, however, is of restricted value due to methodologic difficulties, leading in some cases to false-positive or false-negative results. This study examined the influence of different fixatives and modes of storage of cytologic cell preparations on immunoreactivity using immunoenzymatic techniques. Monoclonal and polyclonal antibodies were used on imprint preparations of breast lesions and tonsils and on bronchoalveolar lavages. A short fixation of the cytologic material with 0.05% glutaraldehyde or a commercially available spray fixative (Merckofix) enabled the immunolocalization of a considerable number of antigens. Optimal demonstration of some antigens, however, required the use of individual fixatives. Drying of cell preparations should be avoided at any step of the procedure to prevent false-positive or false-negative results. Smears stored in methanol or sucrose-MgCl2-glycerol solutions retained their immunoreactivity for several months. The results of this study are compared with those of previous reports, and the reasons for, and means of avoiding, frequent pitfalls in immunocytochemistry are discussed.     

Combined Fixing, Staining and Mounting Media

A number of non-volatile, water-soluble substances can be added to the usual aceto-cannine fixing fluids. These inert substances do not alter the fixation image and serve as mounting media when the volatile ingredients of the mixture evaporate. Formulae are given for solutions containing dextrin, dextrose, gelatin, pectin, sorbitol, and sucrose. Gum arabic can be incorporated in a formic-acid-carmine fixative. The limiting factor in the use of such mounting media in fixing fluids is the osmotic value they give the solution; with certain precautions, however, they can be used in place of the usual aceto-carmine treatment. The indices of refraction of these media are not as high as those of the natural balsams and the fixation images which the mixtures produce have the characteristic limitations of those secured by the aceto-carmine technic. Some of the natural balsams (Canada balsam, sandarac and Venetion turpentine) can also be incorporated in fixing fluids. These fixatives are able to hold balsam and water in solution together, and, as the volatile components of the mixtures evaporate, the fixed specimens remain in permanent balsam mounts. The addition of carmine to these fluids enables us to fix, stain, dehydrate, clear and mount a specimen in a single operation. These fixatives preserve more details of chromosome structure than the aceto-carmiae fluids, but their use is more limited; and they can be substituted for the latter only with certain favorable material, e.g., pollen mother cells of Rhoeo and Tradescantia and salivary gland chromosomes of Cbirono-mus. Some of the newer synthetic resins can be substituted for the natural balsams. Formulae are given for fixatives which contain Venetian turpentine, sandarac, Canada balsam and two synthetic resins.     

Commercial Formalin Substitutes for Histopathology

We compared the performance of six commercial fixatives proposed to be formalin substitutes with the performance of buffered formalin, Clarke's ethanol-acetic acid, and ethanol, using rat liver, small intestine, and kidney. We investigated the rate of penetration, mode of fixation, extent of protein and structural immobilization, quality of histology and cellular structure following routine dehydration and paraffin embedding, and performance as a fixative for immunohistochemistry. Furthermore, we evaluated the effects of the various fixatives on ultrastructure. Only buffered formalin performed equally well on all tissues tested. While several of the commercial fixatives appeared to preserve liver tissue at 200, the preservation of kidney, intestinal villi, and smooth muscle was unacceptable. Histological distortion, cell shrinkage and vacuolization were prominent when the substitutes or ethanol were used. In contrast, these artifacts were found occasionally and to a minor degree when buffered formalin or Clarke's fixative were used. Immunohistochemistry demonstrated a total loss of low molecular weight antigen (serotonin) and patchy reactions for high molecular weight antigens for all fixatives except buffered formalin. The best immunostaining was obtained by combining formalin fixation with antigen retrieval. We conclude that none of the proposed commercial substitutes for buffered formalin are adequate for critical histology or histopathology.     

A Detailed Analysis of the Effects of Various Fixatives on Animal Tissue with Particular Reference to Muscle Tissue

Nine different fixatives (Carnoy's, Susa, Baker's formalin, 5% formalin, 10% formalin, 10% formol saline, Bouin, Zenker, and 2.5% glutaraldehyde) were compared by two methods. Gelatin-albumin gels were used to study volum changes after fixation and after various stages of subsequent processing. The appearance and hardness of the gels were also noted. The fixatives either shrunk or swelled the gels, but dehydration and clearing shrunk the gels in all cases. Sampkes of muscle tissue from one location in beef longissimus dorsi muscle were also placed in the different fixatives and processed. Various features were noted for each fixative, including the ease with which the paraffin wax blocks were cut and the staining ability of the sections in Mallory's triple stain. The diameters of the muscle fibers were measured from transverse sections of these samples and compared with the mean diameter of muscle fibera in a frozen unfixed section of muscle tissue. It was found that the fixatives had the same shrinkage effects on both the gels and the muscle samples. Analysis of variance tests showed that the various fuatives caused different degrees of shrinkage. Statistical details are given for the amounts of shrinkage caused by each fixative. Both the general histological picture and the amount of shrinkage were considered when deciding the bcst fixative. Carnoy was found to be the best of the fixatives investigated.     

Immunofluorescent labelling of K-papovavirus antigens in glycol methacrylate embedded material: a method for studying infected cell populations by fluorescence microscopy and histological staining of adjacent sections

Trypsin and protease V (pronase) were studied for their ability to enhance immunofluorescent labelling of papovavirus antigens in glycol methacrylate embedded sections of organs infected with murine K-papovavirus. Treatment of Bouin's fixed sections with 0.4% trypsin for 30 minutes resulted in specific immunofluorescent staining equal to that seen in frozen sections and produced little if any loss of histological detail. Treatment with protease V resulted in less brilliant fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fixative which would produce brightest specific fluorescent antibody staining of papovavirus-infected cells while providing clearest definition of intranuclear inclusions and best morphological detail in histologically stained adjacent sections. Brightest immunofluorescence staining was accomplished on material fixed in 96% ethanol/1% glacial acetic acid or Bouin's solution. These fixatives also gave clear definition of intranuclear inclusions with histological stains and provided excellent morphological detail. Phosphate buffered paraformaldehyde/picric acid and 3.7% formalin gave less satisfactory fluorescence and obscured intranuclear inclusions in histological preparations. Sections fixed in 4% paraformaldehyde, 4% paraformaldehyde/1% glutaraldehyde, and 0.5 M p-toluenesulfonic acid were negative for specific fluorescence. Glycol methacrylate, used with proper fixation and trypsin pretreatment of sections, provides a useful embedding medium for immunofluorescent identification of virus-infected cells, and the 1.0-2.0 micron sections routinely obtainable with GMA permit study of individual infected cells by fluorescent antibody and histological staining of adjacent sections.     

Tests for Nucleic Acid Selectivity of Einarson's Gallocyanin-Chrome Alum Stain on Monolayers of Strain L-929 Mouse Fibroblasts

L-929 fibroblasts, fixed on coverslips, were stained with gallocyanin-chrome alum after various treatments for removal of nucleic acid or for methylation or deamination. For nucleic acid, trichloroacetic acid and NaCl extractions and sequential incubation in DNase and RNase yielded cells unstainable with the dye complex. Methylated cells showed no cytoplasmic staining and a reduced nuclear staining, compared with the unblocked controls. Deamination had little effect. All results were dependent on the types of fixative used, times and temperatures of incubation, and in the case of nucleases, their concentration. Conventional dehydration and melted paraffin infiltration was associated with little or no staining of deaminated cells and intense staining of methylated cells. The paraffin effects were also dependent on fixatives. The evidence shows that gallocyanin-chrome alum combines with groups (presumably phosphate or carboxyl, or both) which are blocked by methylation, and which can be removed from L cells by sequential RNase and DNase treatment.     

Improved transmission electron microscopy technique for the study of cytologic material

A modification of the technique of Coleman et al for the preparation of single cells in cytologic specimens for electron microscopy (EM) is described. By employing materials in the initial cytologic processing that are useful for EM, such as a paraformaldehyde-glutaraldehyde fixative, lactated Ringer's solution as a rinsing medium, glycerol as a mounting medium and cacodylate buffer for removal of coverslips, the use of alcohol fixatives and standard mounting media could be avoided. This preserved the cytoplasmic detail, which is usually degenerated in cells removed from cytologic specimens and processed for EM.     

An analysis of the contribution of the preparatory technique to the appearance of condensed and orthodox conformations of liver mitochondria

The structure and volume of isolated mitochondria embedded for electron microscopy during different respiratory states were analyzed in thin sections. Three different embedding methods were compared; osmium tetroxide fixation/acetone dehydration, glutaraldehyde fixation/acetone dehydration, and glutaraldehyde fixation-osmium tetroxide postfixation/acetone dehydration. Analysis of fresh mitochondria, isolated in a sucrose medium, revealed the presence of a homogeneous population with respect to structure when any of the three methods were applied. After fixation with osmium alone, or in combination with glutaraldehyde, nearly 100% of the mitochondria were in a "condensed" conformation. Mitochondria fixed with glutaraldehyde alone resulted in a population of mitochondria that had large spaces separating the two membranes of the cristae which corresponds to the condensed conformation as observed after osmium fixation. Transfer of the mitochondria to the incubation medium led to the appearance of two classes of mitochondria with respect to size. One class had a volume close to that observed when suspended in sucrose, and another class was present that was 30-45% larger. In osmium fixed or in double-fixed preparations, these small and large classes corresponded to "condensed" and "orthodox" forms of mitochondria respectively. When glutaraldehyde was used alone as the fixative, the two size classes were also present. However, the mitochondria were homogeneous with respect to structure. In these mitochondria, the width of the space that separated the cristae membranes had become reduced when compared to mitochondria suspended in sucrose. The two size classes were also present in samples of mitochondria prepared during both states 3 and 4. State 4 conditions did not lead to any significant increase of the number of condensed mitochondria. In state 3 preparations, 65-70% of the population were condensed. The condensed and orthodox forms could be related to normal and swollen forms of mitochondria. Conditions that led to a swelling also led to an increase in the number of orthodox mitochondria in osmium-fixed material. The different appearance of the mitochondria is explained by the different conditions for fixation of the mitochondria that exist when nonswollen and swollen mitochondria are fixed. This difference is particularly crucial in the case of osmium tetroxide due to the unique way this fixative, among generally used fixatives, denatures proteins.     

Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and "postfixation" in uranyl acetate

Human leukocytes in suspension or in monolayer cultures have been processed for electron microscopy by fixation in a freshly made cold mixture of glutaraldehyde and osmium tetroxide and by "postfixation" in uranyl acetate. Simultaneous exposure to glutaraldehyde and osmium tetroxide eliminates many of the shortcomings seen when either of these agents is used alone as the initial fixative. Specimens are processed to the stage of dehydration as single cell suspensions or as very small clumps to assure rapid penetration of fixatives and efficient washing. The technique is rapid and reproducible. Electron micrographs presented in this report illustrate the ultrastructural features of human white cells prepared by this method.     

Organization of the cytoskeleton in resting, discoid platelets: preservation of actin filaments by a modified fixation that prevents osmium damage

This study evaluates the structural organization of the cytoskeleton within unactivated, discoid platelets. Previously, such studies have been difficult to interpret because of the ease with which platelets are stimulated, the sensitivity of actin filaments to cell extraction buffers, and the general problem of preserving actin filaments with conventional fixatives, compounded by the density of the cytoplasm in the platelet. In this study we have employed a new fixative containing lysine, which protects actin filaments against damage during fixation and thin-section processing. We used thick (0.25-micron) sections and conventional thin sections of extracted cells (fixed and lysed simultaneously by the addition of 1% Triton X-100 to the initial fixative) as well as thin sections of whole cells to examine three preparations of human platelets: discoid platelets washed by sedimentation; discoid platelets isolated by gel filtration; and circulating platelets collected by dripping blood directly from a vein into fixative. In all of these preparations, long, interwoven actin filaments were observed within the platelet and were particularly concentrated beneath the plasma membrane. These filaments appeared to be linked at irregular intervals to the membrane and to each other via short, approximately 20- to 50-nm-long cross-links of variable width. Although most filaments were outside the circumferential band of microtubules and the cisternae of the open canalicular system, individual filaments dipped down into the cytoplasm and were found between the microtubules and in association with other membranes. The ease with which single actin filaments can be seen in the dense cytoplasm of the human platelet after lysine/aldehyde fixation suggests the great potential of this new fixative for other cells.     

Fixatives and fixation: a review

Conclusions During the period under review, various new fixatives have been introduced. The most important of these is glutaraldehyde which has become very widely used. The processes involved in fixation are becoming better understood in chemical and physical terms, largely due to developments in biochemistry and tanning. This understanding will become increasingly important as the resolution of macromolecules by the electron microscope increases. The images seen will then be able to be interpreted in the light of biochemical knowledge of the macromolecules and thus the gap between morphology and biochemistry will be bridged.Some of the fixatives have secondary uses in that they may demonstrate chemical substances. Others apparently destroy certain biological activities selectively.Increasing attention is being paid to the loss of substances from biological material during preparation. The importance of such studies cannot be overemphasized.No one fixative is ideal for all situations. By comparing the results obtained by a number of fixatives, and assessing the loss of substances during processing, it is far less likely that artefacts will be described as constant features.     

An improved procedure for the histochemical demonstration of cathepsin D by the mercury-labeled pepstatin method

The desirable fixation conditions for the histochemical demonstration of cathepsin D using mercury-labeled pepstatin as an enzyme inhibitor were examined biochemically and histochemically. Four well known fixatives, namely, glutaraldehyde (GA), paraformaldehyde (PFA), glutaraldehyde with paraformaldehyde (GA-PFA) and periodate-lysine-paraformaldehyde (PLP), were applied to the prefixation of tissues prior to the reaction of the labeled inhibitor to the enzyme-active site. The effects of fixatives on cathepsin D were biochemically examined using subcellular fractionated lysosomes. Cathepsin D from rat liver lysosomes was rapidly inactivated by the fixatives containing glutaraldehyde, i.e., GA and GA-PFA, whereas the activity of cathepsin D was sufficiently maintained after fixing the enzyme in the PFA or PLP preparations. Effects of the PLP fixative on lysosomal cathepsin D in liver tissues using the mercury-labeled pepstatin method were also studied histochemically. The best result for the visualization of lysosomal cathepsin D in liver tissues was obtained using the PLP fixative with the prefixation time of three hours or more.     

The effect of digitonin-containing fixatives on the retention of free cholesterol and cholesterol esters

Synopsis The influence of several fixation and dehydration procedures on the retention of free cholesterol and cholesterol esters was studied in filter paper preparations. The retention of free cholesterol by the filter paper proved to be decreased by the addition of digitonin to the aldehyde fixative (aqueous phase) and was only slightly enhanced by partial dehydration (alcoholic phase, up to 70% ethanol). Furthermore, digitonin or the presumably formed cholesterol-digitonide complex bound hardly any osmium oxides in glass-fibre paper.Up to 26% of the cholesterol esters was mobilized during the aqueous phase when digitonin was added to the aldehyde fixative. When the glass-fibre papers containing the digitonin cholesterol-ester-osmate complexes were stored in distilled water after fixation, the fluid became turbid. Particulate material isolated from this turbid solution showed ultrastructurally a close resemblance to the whorls observed by several authors in tissue fixed by a digitonin-containing aldehyde fixative.Digitonin also changed the ultrastructural appearance of liposomes, containing lecithin: cholesterol: phosphatidic acid, in a molar ratio 721. Our observations lead to the conclusion that the use of digitonin-containing fixatives should be abandoned, because they give results which cannot be interpreted. By the use of K₄[Fe(CN)₆] containing OsO₄ in the post-fixation step we were able to demonstrate an increase in the visualization of membranous structures (liposomes).     

A Glycine-Hcl-Formalin Fixative for Improved Staining of Neuroglia

The pH of fixatives as well as the components of the fixative mixture exert a displacing action on the isoelectric point of tissue proteins, which influences the intensity of staining. Studies of these effects showed that ammonium nitrate, sulfate or chloride could be substituted successfully for ammonium bromide in Cajal's formalin-ammonium bromide fixative. However, the best results from staining with Rio Hortega's silver carbonate and with Cajal's gold-sublimate methods were obtained after fixation in a mixture consisting of: glycine, 1.05 gm; 12V HCl, 14.8 ml; concentrated formalin, 15 ml and distilled water to make 100 ml.     

Glutaraldehyde fixative tonicity for scanning electron microscopy of delicate chick embryos.

Fresh pullet eggs (White Leghorn strain) were incubated from 19-2ldium-gold and observed in a Cambridge S4 scanning electron microscope. Shrunken cells with intracellular yolk granules embossed on the surface are produced by the strongly hypertonic Karnovsky's fixer (Final: 2010 mOsm). Embryos fixed with modified Karnovsky's fixer (Final: 373 mOsm) possess surfaces with irregular microappendages. Swollen cells with few microappendages are observed when embryos are fixed in a hypotonic environment (Final: 250 mOsm or less). Ideal fixatives preserve a relatively flat surface, with cells bordered by smoothsurface microappendages. For adequate SEM fixation, fixative vehicle should be approximately isotonic for tissue, with aldehyde (2% or less) added to vehicle.